Trigonelline as a muscle stimulant

ABSTRACT

This invention relates to the use of trigonelline compounds to increase muscle weight during periods of activity or to inhibit muscle loss during periods of inactivity.

This application is a continuation-in-part of PCT patent applicationPCT/EP2010/058124, filed Jun. 10, 2010, and claims priority to EuropeanPatent Applications EP0916247.0, filed Jun. 11, 2009, and EuropeanPatent Application EP09166271.8 filed Jul. 23, 2009, each of which ishereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the use of trigonelline compounds to increasemuscle weight during periods of activity or to inhibit muscle lossduring periods of inactivity.

BACKGROUND OF THE INVENTION

One of the most characteristic features of skeletal muscle is itsremarkable ability to adapt to different stimuli. Throughout life theskeletal muscle is permanently adapting to internal (ageing/sarcopeniaresulting in muscle loss) or external changes (physical activity resultsin muscle hypertrophy; while injury results in muscle recovery; and bedrest results in muscle atrophy). These influences modify structural,biochemical and molecular variables of the different skeletal musclefibers. For the adaptation of myofibers the activation and myogenicdifferentiation of satellite cells, the so-called stem cells of theskeletal muscle, are required. After exercise training, for example,satellite cells fuse together with the enlarging or repairing myofibers.

Trigonelline is an alkaloid with chemical formula C₇H₇NO₂. Trigonellineis a niacin (vitamin B3) metabolite which is excreted in the urine.Trigonelline is also found in coffee, where it may help to preventdental caries by preventing the bacteria Streptococcus mutans fromadhering to teeth.

US 2005/0226948 (Lee et al) discloses a Fenugreek seed extractcontaining 4-hydroxyisoleucine and a number of other compounds,including trigonelline. These combinations are used to enhance glucosetransport into muscle cells.

US2007/0105793 (Hendrix) discloses a composition useful for treatment ofhyperlipidemia, hypercholesterolemia and hyperglyceridemia that containsniacin and derivatives.

US2007/0259861 (Krantz) discloses compositions containing anon-steroidal anti-inflammatory drug (NSAID) in combination with aprostaglandin mimetic (which can include various niacin derivatives.These combinations are used for pain and/or inflammation relief.

DESCRIPTION OF THE INVENTION

It has been found, in accordance with this invention that trigonellinecan increase muscle weight during a training period, and can reducemuscle loss during periods of lesser activity or immobility. Thus, oneaspect of this invention is the use of trigonelline, and/or a salt orester thereof, in the manufacture of a nutraceutical or food to increasemuscle weight during training, or to reduce the amount of muscle lossduring inactivity, with the proviso that if the trigonelline is presentin a plant extract, then the extract is free from tropane alkaloids.Another aspect of this invention is a method of increasing muscle massduring exercise comprising administering an effective amount oftrigonelline or salt or ester thereof to an individual undergoingtraining and observing a muscle mass increase, proviso that if thetrigonelline is present in a plant extract, then the extract is freefrom tropane alkaloids. A further aspect of this invention is a methodof reducing the muscle loss of a less active or immobile person at riskof muscle loss by administering trigonelline or a salt or ester thereof,and observing retention of muscle. In preferred embodiments, thetrigonelline and/or salt or ester thereof is used along with an optimalnutritional supply of protein and vitamins (including especially VitaminD and/or its metabolites such as 25-hydroxyvitamin D3).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of in vitro effects of trigonelline on myotubeformation in mouse muscle cells.

FIG. 2 shows wet weight of the gastrocnemius muscle relative to bodyweight (BW) from control and treated mice (hypertrophy model).

FIG. 3 shows results of the tail suspension model gastrocnemius musclewet weight relative to BW for control and treated mice.

FIG. 4 shows wet weight of gastrocnemius muscle after treadmill running.

As used throughout the specification and claims, the followingdefinitions apply:

“Trigonelline” refers to any form of trigonelline suitable for anutraceutical, including trigonelline, and trigonelline hydrate of atrigonelline salt or ester.

“Trigonelline salt or ester” refers to any salt or ester form oftrigonelline which can be used to formulate trigonelline into anutraceutically acceptable form, or into a foodstuff. The salt or estershould be one which is allowable for ingestion by the applicableregulatory agency. Examples of suitable trigonelline salts includesulfate, hydrogen sulfate, chloride, phosphate, and citrate. Examples ofsuitable trigonelline esters include the methyl ester chloride, andethyl ester chloride.

“Prevent” includes reduction of severity of symptoms and or conditions,reducing the risk of developing a symptom or condition, increasing thetime before symptoms or conditions develop, early intervention, as wellas negation of a symptom or condition.

“Observing” can be done either by the individual who uses thetrigonelline or by a third party. The observation may be done over aperiod of time, such as for one week, one month, three months, sixmonths or other desired time period.

Trigonelline can be found in a large variety of plants such as green androasted coffee beans, Trigonella foenum graecum (fenugreek,Leguminosae), Schumanniophyton magnificum, (Rubiaceae) Mappia foetida,and Strophanthus spp., to name just a few. Thus, trigonelline may bepresent in a plant extract. Preferably, if using a plant extract as thesource of trigonelline, the plant extract contains at least about 20 wt% of trigonelline and the extract is free from tropane alkaloids.

Alternatively, trigonelline can be made synthetically from nicotinicacid. An example of a synthesis is DE 344030 (1921) “Betaines of thepyridine series” (Merck, E.).

It was found that trigonelline can boost the weight of muscles when usedin combination with exercise, and that it can delay the onset of muscleatrophy when muscle is not being used or not being used strenuously.

It is also preferred that if a plant extract is used as the source oftrigonelline, then the plant extract is a coffee bean extract, and theamount of trigonelline is at least 10% of the extract by weight.

It is preferred that the trigonelline is used as part of a nutritionallycomplete diet, i.e. used in conjunction with an adequate or optimalsupply of protein and/or vitamins. Preferably the vitamins will includeVitamin D and/or an active Vitamin D metabolite such as25-hydroxyvitamin D3. Combinations of trigonelline and/or a salt orester thereof along with protein and/or vitamin(s) are also a part ofthis invention.

As trigonelline has applications in veterinary medicine as well as humanmedicine, another aspect of this invention is the use of trigonelline toimprove muscle health in non-human animals, particularly racing horses,dogs, camels or other animals used for racing or as pack animals, orother animals used for their strength. The trigonelline can beadministered to healthy animals, or to injured/sick animals to speedtheir convalescence.

Trigonelline or a salt or ester thereof, without introducing calories tothe diet, has these specific benefits:

-   -   Helps to prevent muscle loss    -   Supports healthy muscle function together with exercise    -   Helps to prevent sarcopenia (risk reduction, reduce severity,        delay progression)    -   Helps to prevent muscle loss during illness or after surgery,        thus contributing to faster convalescence and shorter hospital        stays    -   Helps to prevent frailty in elderly, thus contributing to        improved mobility, quality of life and helps to postpone loss of        independent living    -   Helps one retain muscle development when circumstances prevent        one from exercising    -   Supports an efficient exercise program    -   Supports the efficacy of resistance exercise programs such as        bodybuilding or weight training    -   Supports recovery from muscle damage    -   Helps one retain exercise success/training effects longer    -   Helps one maintain one's shape/condition longer    -   Helps you to find the physical power you need    -   Helps to maintain muscle strength    -   Helps to increase muscle strength    -   Improves body composition together with exercise    -   Supports body toning and body shaping    -   Promotes myoblast differentiation    -   Promotes muscle differentiation    -   Promotes muscle growth    -   Promotes muscle formation    -   Promotes muscle recovery and repair    -   Promotes muscle hypertrophy, when combined with exercise

We surprisingly found that trigonelline helps when the skeletal muscleis adapting to stimuli like training—muscle load (hypertrophy) or tounloading (atrophy). It helps to strengthen the effects of training andprevents skeletal muscle loss. Therefore trigonelline supports anefficient training program and promotes muscle hypertrophy when combinedwith exercise/muscle loading. These effects can be observed.

Thus trigonelline helps to prevent sarcopenia, frailty in elderly, andmuscle loss during bed rest due to illness, surgery and longer hospitalstays. Furthermore, trigonelline promotes muscle recovery and repair.

Dosages:

While dosages may vary, they may range from at least 5 mg per day for ahuman; preferably from 5 to 5,000 mg/day for a human, more preferablyfrom 10 to 3000 mg/day for a human and even more preferably from 50-500mg/day for a human. Other preferred dosages include:

-   -   50-100 mg/day    -   75-125 mg/day    -   100-150 mg/day    -   125-175 mg/day    -   200-225 mg/day and    -   250-300 mg/day.    -   Animal dosages are similar, and can be adjusted accordingly for        the weight of the animal.

In accordance with this invention, the trigonelline or salt or esterthereof is present in a dietary, nutraceutical, or pharmaceuticalcomposition. Preferred compositions comprise trigonelline or a salt orester thereof and a suitable dietary, nutraceutical or pharmaceuticalcarrier. The dietary products or nutraceuticals of this invention can bein any format acceptable to the consumer, including functional food andbeverages.

Examples of suitable nutritional formats include various foods andbeverages, including shots, cereal or other bars, drinks, protein-richdrinks, supplements, instant beverages, effervescents and the like.Especially preferred are formats which are suitable for sportsnutrition, including beverages, protein powders, bars, supplements andinstant beverages.

The following non-limiting Examples are presented to better illustratethe invention.

EXAMPLE 1 Muscle Cell Model

We first tested the influence of trigonelline on myoblastdifferentiation in vitro using C2C12 mouse myoblast cells which arecommonly used to study muscle adaptation. C2C12 cells were seeded on 96well collagen-I plates (1600 cells/well) in growth medium (Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% FBS, 2 mML-Glutamine, 1 mM pyruvate, 50 IU/ml penicillin, 50 ug/ml streptavidin)until it reaches 100% confluency, approximately 3 days. Cells were theninduced to differentiate using differentiation medium (DMEM, 2% FBS, 2mM L-Glutamine, 1 mM pyruvate, 50 IU/ml penicillin, 50 ug/mlstreptavidin) with either DMSO (control) or 10 μg/ml trigonellinemonohydrate for 24 h. Final DMSO concentration was standardized to 0.5%in all wells. Cells were cultured at 37° C., with 5% CO₂.

Cells were fixed with 3.7% formaldehyde solution (37% formaldehydediluted in growth medium, sterile filtered before used) for 10 min. atroom temperature, and washed twice with 1×DPBS (1× Dulbecco's PhosphateBuffered Saline). After permeabilization with 0.1% TritonX-100 (dilutedin 1×DPBS) for 2 min. the samples were blocked with 1×DPBS plus 2% BSAfor 1 h. For myocyte labelling, the cells were labelled with 1:20anti-alpha-myosin heavy chain antibody (MF-20) for 1 hour. The sampleswere washed with 1×DPBS and fluorescently labelled with secondaryantibody (1:250 Alexa 488 IgG antimouse secondary antibody plus 1:2000Hoechst in 1×DPBS) for 1 h. Finally, the cells were washed twice with1×DPBS and plates were sealed for imaging on the ArrayScan® HCS Reader.

For image analysis myosin labelled myotubes were quantified on theArrayScan HCS Reader using BioApplication Morphology Explorer.V2.Myocytes were selected with Object Area larger than 500 and Object TotalIntensity greater than 1000. The number of myocytes from triplicatesamples are shown in FIG. 1. Trigonelline was seen to increase thedifferentiation in C2C12 mouse myoblast cells by 36%.

EXAMPLE 2 In Vivo Effects

To confirm our in vitro results, trigonelline was first tested in ahypertrophy animal model to see the effects during muscle load. Thegastrocnemius muscle from one hindlimb of the animals was removed toinduce compensatory hypertrophy in the plantaris and soleus muscles bymultiple mechanisms. This model increases the muscle weight under muscleload/training. It simulates the human condition of an average human whois physically active or an athlete to support skeletal muscle functionduring exercise.

Female C57Bl/6 mice were delivered at a weight of 18-20 g andacclimatized to the facilities for a period of one week. At thebeginning of the study the animals were randomized into two groups (10animals per group).

The animals were anesthetized and the left hindlimb of the animals wasfixed. A small incision was made through the skin over the gastrocnemiusmuscle. The complete gastrocnemius muscle and its tendons were exposed.Both heads of the gastrocnemius muscle were carefully dissected from theunderlying intact muscles and care was taken not to rupture nerves andvessels. The skin was closed with a silk and the animals were returnedseparately into their cages. After recovering from anesthesia, theanimals could move immediately without problems. All animals received ananalgesic. Animals were treated for three weeks by gavage withtrigonelline-hydrochloride at a daily dosage of 300 mg/kg BW and thecontrol group received vehicle.

Using this technique we have identified and quantified increasedskeletal muscle weight of the plantaris and soleus muscle in theoperated (pQCT-measurement) and in the gastrocnemius, plantaris andsoleus muscle of the non-operated leg (weight measurement). Thehypertrophy in the operated leg is a compensatory reaction of theremaining muscles after the gastrocnemius muscle has been removed. Thehypertrophy on the non-operated leg is due to a special training effect,because this leg was used more.

The wet weight of the non-operated gastrocnemius muscle (relative to thebody weight) increased by 7% in the group receiving trigonelline (FIG.2).

The absolute and the relative (compared to body weight of the animals)wet weight of all analyzed muscles from the non-operated leg wereincreased.

In the operated leg, the total leg weight and the muscle cross-sectionalarea, as assessed using computer-tomography measurement were alsoincreased. Data are shown in Table 1.

TABLE 1 Trigonelline- hydrochloride Control 300 mg/kg/BW Parameters (n =10) (n = 10) M. gastrocnemius wet weight (mg) 98.5 99.3 (non-operatedleg) M. plantaris wet weight (mg) 12.4 13 (non-operated leg) M. soleuswet weight (mg) 7.1 7.2 (non-operated leg) M. gastrocnemius wetweight/body 4.44 4.57 weight (mg/g) (non-operated leg) M. plantaris wetweight/body weight 0.56 0.60 (mg/g) (non-operated leg) M. soleus wetweight/body weight 0.32 0.33 (mg/g) (non-operated leg)Gastrocnemius-Plantaris-Soleus 5.32 5.50 muscle weight (whole leg)/bodyweight (mg/g) (non-operatedleg) Total leg cross-sectional area (mm²)-33.90 35.05 non operated leg Muscle cross-sectional area (mm²)- 26.2732.15 non operated leg Total leg cross-sectional area (mm²)- 25.40 26.79operated leg Muscle cross-sectional area (mm²)- 19.13 23.72 operated leg

These results show that under loading (after surgery of thegastrocnemius the muscle-hypertrophy)/training conditions, skeletalmuscle wet weight increased in the animals.

EXAMPLE 3 Muscle Atrophy

To extend our in vitro and in vivo results we were also interested intesting the effects of trigonelline during skeletal muscle unloading(atrophy conditions). Therefore we conducted a second in vivo study werethe hindlimbs of the animals were unloaded to induce skeletal muscleatrophy.

Tail suspension leads to skeletal muscle atrophy in the unloadedhindlimbs of the animals. The results can be transferred to the humansituation: sarcopenia (degenerative loss of skeletal muscle mass andstrength during the process of ageing) or immobilization of skeletalmuscle (e.g. after prolonged bed rest).

Female C57Bl/6 mice were delivered at a weight of 18-20 g andacclimatized to the facilities for a period of one week. At thebeginning of the study the animals were randomized into two groups (10animals per group).

Thereafter the groups were placed in special cages and the hindlimbswere unloaded (tail suspension) for a duration of three weeks. All micewere housed separately and had access to feed and water ad libidum.Animals were treated for three weeks by gavage withtrigonelline-hydrochloride at a daily dosage of 300 mg/kg/BW and thecontrol group received vehicle.

We found that the wet weight of the gastrocnemius muscle (relative tothe body weight) increased by 9% (FIG. 3), a statistically significantresult.

We also compared the other two muscles from the operated hindlimb(plantaris muscle and soleus muscle). The results, presented in TABLE 2below, show an increase of the wet weights of the muscles in animalstreated with trigonelline-hydrochloride compared to the control animals(absolute muscle weight). Furthermore, when muscle weights werenormalized to the body weights, animals treated withtrigonelline-hydrochloride demonstrated an increased muscle weight/bodyweight ratio (relative muscle weight), i.e, an improved bodycomposition. The total weight for muscles in one leg was alsosignificantly increased when treated with trigonelline-hydrochloride(whole leg relative to body weight). Computer tomography measurements oftotal and muscle leg area confirmed that trigonelline-hydrochloridetreatment increases skeletal muscle mass.

TABLE 2 Trigonelline- hydrochloride Control 300 mg/kg/BW Parameters (n =10) (n = 10) M. gastrocnemius wet weight (mg) 78.13 86.01 * M. plantariswet weight (mg) 9.84 10.53 M. soleus wet weight (mg) 4.29 4.62 M.gastrocnemius wet weight/body 3.83 4.16 * weight (mg/g) M. plantaris wetweight/body 0.48 0.51 * weight (mg/g) M. soleus wet weight/body 0.210.22 weight (mg/g) Gastrocnemius-Plantaris-Soleus 4.52 4.89 * muscleweight (whole leg)/body weight (mg/g) Total leg cross-sectional area(mm²) 29.1 30.4 Muscle cross-sectional area (mm²) 26.6 27.8 *significant: p ≦ 0.05

The results show that under unloading/atrophy of the animal total legcross-sectional area, the muscle weights increase; i.e. more of themuscle mass is retained during inactivity in trigonelline-supplementedanimals versus controls.

EXAMPLE 4 Endurance

To test the effect of trigonelline in non-trained animals, we performeda maximal endurance test after a treatment period of three weeks.

Female C57Bl/6 mice were delivered at a weight of 18-20 g andacclimatized to the facilities for a period of one week. At thebeginning of the study the animals were randomized into two groups (10animals per group). All mice were housed separately and had access tofeed and water ad libidum. Animals were treated for three weeks bygavage with trigonelline-hydrochloride at a daily dosage of 300 mg/kg/BWand the control group received vehicle.

For acclimatization, animals were placed on the treadmill for 5 minutesafter two weeks. The maximal endurance test was performed two day beforethe section of the animals.

We found that the wet weight of the gastrocnemius muscle was equal inboth groups (FIG. 4), as was the wet weight of the other musclesassessed (data not shown). Also, endurance of untrained animals was notinfluenced by 3 weeks of trigonelline supplementation.

Trigonelline- hydrochloride Control 300 mg/kg/BW Parameters (n = 10) (n= 10) Mean running time (minutes) 39.00 38.70 M. gastrocnemius wetweight (mg) 100.65 100.66 M. plantaris wet weight (mg) 12.16 12.12 M.soleus wet weight (mg) 6.28 6.29 M. gastrocnemius wet weight/ 4.64 4.57body weight (mg/g) M. plantaris wet weight/body 0.56 0.55 weight (mg/g)M. soleus wet weight/body 0.29 0.29 weight (mg/g)Gastrocnemius-Plantaris-Soleus 5.49 5.41 muscle weight (whole leg)/bodyweight (mg/g)

These results show that trigonelline has no effect on endurance andskeletal muscle mass in untrained mice.

1. A method of increasing muscle mass in combination with an exerciseselected from the group consisting of resistance training, bodybuildingor weight training in a human in need thereof consisting essentially ofadministering therapeutically effective amounts of a coffee bean extractwhich consists of at least 20 wt. % of trigonelline and25-hydroxyvitamin D3 with the proviso that the extract is free fromtropane alkaloids to said human.